Project Summary/Abstract Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex disorder characterized by profound fatigue, as well as immune and neurocognitive dysfunction. The etiologies and the drivers of ME/CFS remain unknown and there are no useful biomarkers for distinguishing the various subgroups. Our innovative studies over the ten previous years of this award have demonstrated that the deoxyuridine triphosphate nucleotidohydrolases (dUTPase) encoded by the Epstein-Barr virus (EBV) and more recently by the human herpesvirus 6 (HHV-6) have novel immunoregulatory and neuroregulatory functions that contribute to the pathophysiology observed in a subgroup of patients with ME/CFS. In this renewal application, we will continue our mechanistic studies to define the role that the EBV and HHV-6 dUTPases have in ME/CFS. Our overall hypothesis is that the increased abortive replication of EBV in plasmablasts/plasma cells in a subgroup of patients genetically susceptible to developing ME/CFS induces the increased synthesis and release of EBV dUTPase in exosomes. Ligation of EBV dUTPase-containing exosomes with TLR2 present on antigen- presenting cells (APCs) in the local microenvironment triggers the production of pro-inflammatory TH1 cytokines as well as activin A, which in turn stimulates the differentiation and proliferation of follicular helper T (TFH) cells, resulting in IL-21 production, which contributes to the immune dysfunction observed in these patients. Activin A is also a negative regulator of muscle growth and we are proposing that it alters the function of key enzymes in skeletal muscle leading to oxidative stress and low energy levels thus, contributing to the post-exertional fatigue characteristic of patients with ME/CFS. Trafficking of EBV dUTPase containing exosomes to the brain and TLR2 ligation on target cells results in microglia activation, disruption of the BBB, neuroinflammation and altered synaptic plasticity ultimately leading to cognitive impairment in these patients. Finally, these physiological effects of the EBV dUTPase are enhanced by chronic stress. The results of this study, will demonstrate that the EBV/HHV-6 dUTPases may act as drivers of the immune activation that occurs in ME/CFS. It may also identify EBV/HHV-6 dUTPase proteins as diagnostic biomarkers for identifying a subset of patients with ME/CFS. Finally, these studies will demonstrate that engagement of EBV/HHV-6 dUTPase proteins with TLR2 is essential for inducing neuroimmune dysfunction and thus, this interaction can be used as a novel target for the development of alternative therapeutic approaches.